Licorice root extracts have been extensively used for their therapeutic properties which include the potentiation of cortisol action, inhibition of testosterone biosynthesis, reduction in body fat mass and other endocrine effects (1-4). The activities of these extracts are linked to different classes of phytochemicals particularly the major water soluble constituent glycyrrhizin and its hydrolysis product 18β-glycyrrhetinic acid (GA):
Glycyrrhizin is a pentacyclic triterpenoid glycoside which is hydrolyzed in the gut to GA and many of the properties of licorice root can be attributed to GA. For example, GA inhibits 11β-hydroxysteroid dehydrogenase activity increasing corticosterone levels and this has been linked to apoptosis in murine thymocytes, splenocytes and decreased body fat index in human studies (5-9). GA also directly acts on mitochondria to induce apoptosis through increased mitochondrial swelling, loss of mitochondrial membrane potential and release of cytochrome C (10, 11).
GA has also been used as a template to synthesize bioactive drugs. For example carbenoxolone is the 3-hemisuccinate derivative of GA and this compound has been used for the treatment of gastritis and ulcers (12). Some of the activity of carbenoxolone may be due to hydrolysis to GA however carbenoxolone itself induced oxidative stress in liver mitochondria and decreased mitochondrial membrane potential. Other carboxyl and hydroxyl derivatives of glycyrrhetinic acid inhibit HIV and exhibit anti-inflammatory and immunomodulatory activities (13). In addition, GA derivatives containing a reduced carboxylic acid group (CH2OH) at C-30 and some additional functional changes exhibited strong antioxidant activity (14).
GA is an oleanane derivative and there have been extensive structure-activity studies on the anti-inflammatory activities of oleanolic and ursolic acids derivatives (15-19). Two examples that have been prepared and studied are 2-cyano-3,12-dioxo-oleana-1, 9(11)-diene-28-oic acid (CDDO) and its methyl ester (CDDO-Me) which contain major structural differences in the E-ring compared to GA:
Subsequent studies have demonstrated that CDDO activates peroxisome proliferator-activated receptor γ (PPARγ) (20-22).
PPAR is a member of the nuclear receptor (NR) family of transcription factors (23-27), and the three members of this subfamily serve as regulators of lipid and carbohydrate metabolism and play a critical role in multiple diseases including diabetes, atherosclerosis and cancer. Ligand activation of PPARγ results in formation of a DNA-bound heterodimer with the retinoic acid X receptor (RXR) and after recruitment of the appropriate nuclear factors, transcriptional activation of target gene expression is observed. The assembly of a transcriptionally-active PPAR/RXR complexes may be highly variable and dependent on expression of coregulatory proteins, and this may dictate, in part, the tissue-specific and ligand structure-dependent activation of PPAR-mediated gene expression and responses.
PPARγ agonists have been developed for treatment of metabolic diseases, and thiazolidinediones (TZDs) are PPARγ agonists and are used by millions of patients in the United States for treatment of insulin-resistant Type II diabetes. PPARγ is overexpressed in multiple tumor-types (28), and there is evidence that various structural classes of PPARγ agonists inhibit growth and induce apoptosis in both pancreatic and colon cancer cells and tumors (29-50). However, it is clear from studies with PPARγ agonists that their effects in colon, pancreatic and other cancer cell lines and tumors are highly variable and can be mediated through receptor-dependent and -independent pathways. Nevertheless, this characteristic of multiple mechanisms can be advantageous for cancer chemotherapy by targeting several pathways that inhibit tumor growth and metastasis.
Specificity protein 1 (Sp1) was the first transcription factor identified (51), and the Sp/Krüppel-like factor (KLF) family of zinc finger transcription factors exhibit a broad range of tissue-specific and overlapping functions (52-56). Sp1 and Sp3 proteins are ubiquitously expressed and have been extensively investigated. For example, Sp1−/− embryos exhibit multiple abnormalities, retarded development and embryolethality on day 11 of gestation (57). Sp3−/− mice also exhibit growth retardation, defects in late tooth development, and the animals die at birth (58, 59). The critical requirement for Sp proteins during embryonic and postnatal development is in contrast to decreased expression in mature tissue/organs which are relatively quiescent. In contrast, there is increasing evidence that Sp1 (the major focus of most studies) and other Sp proteins such as Sp3 and Sp4 are overexpressed in tumors compared to most other tissues/organs (60-65). For example, a recent study compared the expression of Sp1, Sp3 and Sp4 in prostate and pancreatic tumors in xenograft or orthotopic mouse models, and results illustrated the high expression in LNCaP prostate tumor xenografts vs. normal mouse liver from the same animals (66, 67). Levels of Sp1, Sp3 and Sp4 expression were barely detectable in liver and other tissues compared to high levels of Sp1, Sp3 and Sp4 in tumors, and several studies report that Sp proteins are overexpressed in multiple tumors (60-65). Lou and coworkers (68) reported that transformation of fibroblasts resulted in an 8- to 18-fold increase in Sp1 expression, and these transformed cells formed highly malignant tumors in athymic nude mouse xenograft models, whereas untransformed fibroblasts expressing low levels of Sp1 did not form tumors. In addition, ribozyme-dependent knockdown of Sp1 in the transformed cells decreased VEGF expression and increased apoptosis. Recent studies in this laboratory using RNA interference and other techniques have demonstrated that knockdown of Sp1, Sp3, Sp4 and their combinations decreases cell cycle progression, increases p27 expression, decreases levels of the antiapoptotic protein survivin, and downregulates expression of VEGF, VEGF receptor 1 (VEGFR1) and VEGFR2 (KDR) (66, 67, 69-72).
Since Sp proteins are overexpressed in tumors/cancer cells and play an important role in regulating expression of growth, angiogenic and survival genes, agents that target Sp protein degradation will be highly effective anticancer drugs. For example, the COX-2 inhibitor celecoxib decreased the expression of Sp1 and VEGF by inducing degradation of Sp1 in pancreatic cancer cells (73), and studies showed that COX-2 inhibitors decrease VEGF expression in colon cancer cells by decreasing the level of Sp1 and Sp3 (69). Further, a series of nonsteroidal anti-inflammatory drugs were screened for activity in decreasing Sp protein expression in pancreatic cancer cells (66). The results showed that only tolfenamic acid and structurally related analogs decreased Sp1, Sp3 and Sp4 expression in Panc1 and L3.6pl pancreatic cancer cells through activation of the proteasome pathway, and this was accompanied by decreased VEGF and VEGFR1 expression, increased apoptosis, and decreased cell growth. Moreover, in an orthotopic model for pancreatic cancer, tolfenamic acid decreased Sp protein expression in tumors, decreased tumor growth, decreased angiogenesis (and VEGF), and inhibited liver metastasis. Similar results were also observed using the triterpenoid natural product betulinic acid using LNCaP prostate cancer cells and tumors in a xenograft model (67). These results demonstrate that drugs that target Sp proteins constitute a highly effective and important class of mechanism-based anticancer drugs.